Application of DNA methyltransferase inhibitor in improvement of animal round sperm injection embryo development efficiency

A methyltransferase and embryo development technology, applied in the field of medicine, can solve the problems of high abortion rate, low live birth rate, low implantation rate and the like

Active Publication Date: 2021-07-30
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although ROSI technology has been applied in a variety of animals including mice, rabbits, monkeys and humans, there are still problems such as low implantation rate, high miscarriage rate, and low live birth rate

Method used

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  • Application of DNA methyltransferase inhibitor in improvement of animal round sperm injection embryo development efficiency
  • Application of DNA methyltransferase inhibitor in improvement of animal round sperm injection embryo development efficiency
  • Application of DNA methyltransferase inhibitor in improvement of animal round sperm injection embryo development efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Effect of DNA methyltransferase inhibitor RG108 on improving the developmental efficiency of round sperm injection embryos

[0065] 1. Oocyte Collection

[0066] (1) Prepare egg storage dishes in advance: use imported Eppendorf embryo culture dishes with a diameter of 3.5cm, make eight 30μL culture drops with M16 medium for each dish, add 2.5mL paraffin oil, submerge the culture medium, and put them in the incubator (37°C, 5% CO 2 ) Pre-balanced for 3h.

[0067] (2) Select 10-week-old B6D2F1 female mice in the estrous period (F1 offspring obtained by mating DBA / 2N male mice with C57BL / 6N female mice, C57BL / 6N, DBA / 2N purchased from Beijing Weitong Lihua), The weight is about 18-25g. Calculate the unit of hormone injection according to body weight, inject 8U per 20g of body weight; inject pregnant horse serum gonadotropin (PMSG) intraperitoneally at around 7:30 in the evening of the same day, and inject human chorionic gonadotropin 48 hours after PMSG starts...

Embodiment 2

[0090] Example 2 Effect of the Concentration and Action Time of DNA Methyltransferase Inhibitors on the Blastocyst Development Rate of Round Sperm Injection Embryo

[0091]1. The effect of the concentration of DNA methyltransferase inhibitor RG108 on the blastocyst development rate of round sperm injection embryos

[0092] This test process is identical with step 1, 2, and 3 in embodiment 1, and the difference is only in:

[0093] (1) The concentration of DNA methyltransferase inhibitor RG108 in the activation solution and KSOM culture solution in the third step of the test process was 5 μM, 10 μM, and 20 μM, respectively, and each treatment was repeated 3 times.

[0094] The result is as figure 2 Shown: The concentration of DNA methyltransferase inhibitor RG108 at a concentration of 5 μM and 10 μM can promote the blastocyst development rate of round sperm injection embryos better than the concentration of 20 μM DNA methyltransferase inhibitor RG108 and the control group (DM...

Embodiment 3

[0099] Example 3 Effect of DNA methyltransferase inhibitors on the blastocyst development rate of round sperm injection embryos

[0100] This test process is identical with step 1, 2, and 3 in embodiment 1, and the difference is only in:

[0101] (1) The type and concentration of DNA methyltransferase in the chemical activation solution containing DNA methyltransferase and KSOM culture medium are different during the test: Figure 4 Middle: "ROSI" means without any treatment; "+DMSO" means adding the same amount of DMSO; "+RG108-10μM" means RG108 with a concentration of 10μM; "Thioguanine-2μM" means Thioguanine with a concentration of 2μM (CAS No.: 154 -42-7); "Thioguanine-1μM" means that the concentration is 1μM of Thioguanine; "Mylosar-1.5μM" means that the concentration is 1.5μM of Mylosar (CAS number: 320-67-2); "Mylosar-500nM" means that the concentration is 500nM Mylosar.

[0102] The result is as Figure 4 Shown: DNA methyltransferase inhibitor RG108 can significantl...

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Abstract

The invention belongs to the field of medicines, and discloses application of a DNA methyltransferase inhibitor to improvement of animal round sperm injection embryo development efficiency. The invention discloses the application of the DNA methyltransferase inhibitor in improving the development efficiency of the animal round sperm injection embryo for the first time; compared with an existing round sperm injection technology, the blastocyst development rate of the round sperm injection embryo can be remarkably improved through the DNA methyltransferase inhibitor under the condition that transgenic risks are not introduced; furthermore, excellent round sperm injection embryo in-vitro culture system is established, and the feasibility of a round sperm injection technology is improved.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to the application of DNA methyltransferase inhibitors in improving the development efficiency of animal round sperm injection embryos. Background technique [0002] There are no mature sperm or elongated sperm cells in the testes and epididymis of patients with severe azoospermia, so they cannot obtain genetic offspring by intracytoplasmic sperm injection (ICSI). The round spermatid injection (ROSI) technology is to inject round sperm into oocytes and fertilize oocytes through auxiliary activation, thereby helping these azoospermic patients obtain genetic offspring. Although the ROSI technique has been applied in a variety of animals including mice, rabbits, monkeys, and humans, there are still problems such as low implantation rate, high abortion rate, and low live birth rate. Therefore, improving the developmental potential of ROSI embryos can strongly promote the clinical ap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/075C12N5/076
CPCC12N5/0604C12N5/0609C12N5/061C12N2501/72C12N2500/12C12N2500/16C12N2500/20C12N2501/999
Inventor 赵小阳王京任少芳周偲马琳子李朝晖罗芳常港
Owner SOUTHERN MEDICAL UNIVERSITY
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